Project 1, Miller: Novel Functions of the Epstein-Barr Viral Lytic Cycle Activator Protein ZEBRA

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Project 1, Summary/Abstract Epstein-Barr virus (EBV) is a human oncogenic virus associated with carcinomas of global importance, such as gastric and nasopharyngeal cancer, several lymphomas, including Burkitt, Hodgkin and diffuse large cell lymphoma that occur in immunocompetent individuals and polyclonal lymphomas in immunodeficient hosts. Our laboratory focuses on the EBV lytic cycle that is essential for transmission of virus among cells and between individuals and directly contributes to viral oncogenicity. The EBV lytic cycle is initiated by expression of two viral proteins, ZEBRA and Rta, transcription factors that also play distinct direct roles in lytic viral DNA replication. Our proposed work is based on newly recognized functions of ZEBRA, the major lytic cycle activator protein originally discovered in our laboratory. In AIM1, in collaboration with the Steitz lab, we will explore mechanisms underlying the novel role of ZEBRA in mediating viral host shut off of cellular gene expression. We will investigate how ZEBRA controls the translocation of cytoplasmic poly A binding protein (PABPC) to the nucleus, how ZEBRA selectively distributes PABPC within the nucleus, how ZEBRA retains poly-adenylated mRNA in the nucleus and how ZEBRA selectively inhibits synthesis of cellular but not viral proteins. In AIM2, together with the lab of Yong Xiong, we will study the structural basis of preferential recognition of methylated DNA by ZEBRA. We will investigate the methylation haplotype of promoters of viral and cellular genes targeted by ZEBRA. We will uncover features of the methylated ZEBRA response elements in viral and cellular DNA and properties of the ZEBRA protein itself that mediate preferential recognition of methylated DNA. These studies will eventually yield a crystal structure of ZEBRA bound to methylated DNA. Our proposed experiments will elucidate molecular mechansims that govern two previously unknown functions of the major EBV lytic cycle activator protein, namely viral host shutoff and transcriptional activation via recognition of methylated DNA. These proposed studies have particular relevance for EBV-positive gastric cancer where the presence of the EBV genome induces a state of cellular gene hypermethylation.